The technique of flow cytometry provides investigators with data on physical parameters of cells which can be used to separate various classes of cells, e.g., to classify white blood cells (leukocytes) into three major groups, namely, lymphocytes, monocytes and granulocytes. Other techniques have been described to identify additional leukocyte populations, for example, T-cells, B-cells, cytotoxic cells and suppressor cells, neutrophil, eosinophil, and basophils. See, e.g., Flow Cytometry and Sorting, page 371; edited by Melamed, Mullaney, and Mendelsohn, 1979, John Wiley & Sons, N.Y., N.Y.; U.S. Pat. No. 5,125,737; U.S. Pat. No. 5,492,833; U.S. Pat. No. 5,223,398; U.S. Pat. No. 5,231,005.
The enumeration of subclasses using antibody-coated polystyrene latex beads to target specific white blood cell subpopulations in whole blood, hence, producing a change (a shift) in the targeted cell volume, direct or low frequency current (DC) or high frequency (RF) conductivity, and light scatter (S) has been described. See, e.g., PCT Publication WO92/09682 published Jun. 11, 1992; J. C. Hudson et al, Cytometry, 22:150 (1995), U.S. Pat. No. 5,639,620 and International Patent application No. WO95/24631, published Sep. 14, 1995. In fact, these latter changes can be detected on the commercially available hematology instruments, such as the COULTER.RTM. STKS instrument equipped with VCS technology. Such instruments can calculate the percentage of shifted cell population in the total population of white blood cells.
All of the above bead-induced shift methods use a single type of polystyrene bead conjugated to an antibody to enumerate subpopulations of lymphocytes one at a time. The use of beads composed of inorganic materials to extend shifts has only recently been described. See, e.g., U.S. Pat. No. 5,466,609; International Patent Application No. WO9524631; and U.S. Pat. No. 5,552,086. Specifically, gold/silver colloid coated polystyrene-aminodextran beads, their preparation and characterization have been described. See, e.g., U.S. Pat. No. 5,248,772 and U.S. Pat. No. 5,552,086.
Current methods of obtaining lymphocyte differentials involve the use of fluorescence markers and measurement of forward light scatter, side light scatter and fluorescence intensity or the use of antibody on beads and measurement of median angle light scatter, DC, and RF. Heretofore, the use of antibody on beads has involved only a single type of bead coated with antibody at any one time to analyze for a single subpopulation of lymphocytes. See also, P. K. Horan and L. L. Wheeless, Jr., Science, 198:149-157 (1977); and British Patent No. 1561042, published Feb. 13, 1980. On the other hand, conventionally practiced flow cytometry with fluorescence markers simultaneously uses a number of fluorescent-labeled antibodies (2-6) to analyze for various subpopulations of leukocytes.
At present, there is no method that allows a sample of blood to be analyzed using the antibody on bead method, such that subpopulations of lymphocytes may be simultaneously enumerated. Thus, there exists a need in the art for compositions and methods which enable more efficient analysis of blood or other multicellular fluid materials.